Eukaryotic mRNAs have a cap structure at their 5′-termini. The cap consists of 7-methyl guanosine (m7G) and a triphosphate bridge, ppp (p3), linking the 5′OH of m7G to the 5′OH of the 5′-terminal nucleotide, N, denoted m7G(5′)pppN (m7G(5′)p3N). In eukaryotic cells, the cap structure participates in assembly of the translation initiation complex by binding eukaryotic translation initiation factor 4E (eIF-4E).
Although m7G(5′)p3 can be used to initiate transcription with T7 or SP6 DNA-dependent RNA polymerase in vitro, it has the disadvantage of having to compete with guanine nucleotide (G) as the initiating nucleophile for transcriptional elongation. As a result of this competition, less than half of mRNA produced in vitro have a cap structure at their 5′ termini.
Dinucleotide m7G(5′)p3(5′)G, in which a guanine nucleotide (G) is linked via its 5′OH to the triphosphate bridge, has been used as an initiator of transcription. This dinucleotide has the disadvantage that the 3′-OH of either the m7G or G moiety serves as the initiating nucleophile for transcriptional elongation that results in synthesis of two isomeric RNAs of the form m7G(5′)p3G(pN)n and G(5′)p37G(pN)n, with one third to one half of the caps oriented in the reverse direction, depending upon the ionic conditions of the transcription reaction. Further improvement of the orientation of the cap during in vitro transcription is possible using cap analogues that replace the 3′-OH group with hydrogen or —OCH3 (U.S. Pat. No. 7,074,596; Kore, 2006, Nucleotides, Nucleotides, and Nucleic Acids, 25: 307-14, and Kore, 2006, Nucleotides, Nucleotides, and Nucleic Acids, 25: 337-40).
Dinucleotide GG cap analogs m7G(5′)p3G and 3′-OMe-m7G(5′)p3G (ARCA) are sold commercially by TriLink BioTechnology, MilliporeSigma, ThermoFisher Scientific, and New England BioLabs Inc. 3′-OMe-m7G(5′)p3G (ARCA) is incorporated during transcription without reversal to G(5′)p3 m7G.
Trinucleotide cap analogs were disclosed by Ishikawa, 2009, Nucleic Acid Symp. Ser., 53:129-30. These are                m7G(5′)p3ApG, corresponding to the terminal trinucleotide of plant RNA;        m7G(5′)p3AmpG (Am is adenine with a 2′OMe-ribose), corresponding to the terminal trinucleotide of animal RNA;        m7G(5′)p3 m6AmpG (m6A is N6-methyladenine), corresponding to the terminal trinucleotide of mammal RNA; and        m7G(5′)p3 m6ApG, an unnatural trinucleotide.Ishikawa discloses that translational efficiency in a rabbit reticulocyte lysate system is greatest with mRNA transcribed with an animal-type, followed by a mammalian-type, the unnatural, and the plant-type cap structures, respectively, and that G(5′)p3Ampm7G-RNA, a result of transcribing in a reverse orientation, was not obtained. Id.        
In view of the disclosure of these publications, there remains a need to identify modified cap structures that improve the efficiency of in vitro transcription.